Tight control of gene expression is modulated by RNA binding proteins (RBPs). Transitions in post- transcriptional programs during disease and development are driven by rapid changes in the expression level of splicing factors. For example, in myotonic dystrophy type 1 (DM1) increasing disease severity corresponds to the degree of MBNL1 depletion by toxic RNA repeats. Additionally, RBFOX1, along with MBNL, are tissue- specific splicing factors, whose expression also changes significantly during the course of heart development. RBFOX proteins are also important for human embryonic stem cell survival and epithelial to mesenchymal transformation. To tune sensitivity to developmental changes in RBP levels, highly regulated RNA processing events may require moderate affinity binding sites. However, current methods for characterizing RBP preferences fail to detect these moderate affinity targets. This proposal uses high-throughput biophysical and computational techniques to identify features of RNAs targeted by two distinct families of RBPs. Deep biophysical characterization of RBFOX will identify functional elements that regulate stem cell survival and cancer. Results from these experiments will also provide a coherent description of target sequences and RNA structures that MBNL1 recognizes, ultimately providing an enhanced understanding of myotonic dystrophy pathology. The deep biophysical characterization of MBNL1 will unveil temporal transitions in RNA processing that occur with increasing DM severity. We will determine RNA affinity landscapes for MBNL1 and RBFOX to elucidate the role of RNA affinity in development and disease contexts.